The behavior of cholesterol and low density lipoprotein (LDL) is crucial to our understanding of heart and vessel disease (7-11). High levels of LDL-cholesterol (LDL-C) in the plasma indicate a higher risk of heart attacks, atherosclerosis and stroke. Currently, atherosclerosis control is achieved by treatment with commercially available statin drugs, which induce the up-regulation of LDL-receptor (LDL-R) expression via their ability to strongly inhibit HMG-coA reductase (10, 11, 40). However, increasingly lower LDL-C goals, growing target populations and significant variability and toxicity in the response to available LDL-C lowering therapies, have demonstrated the need for novel therapeutic strategies to lower LDL-C plasma levels (11, 31).
LDL-R is essential for cholesterol homeostasis and thus cardiovascular health, since it is involved in the cellular uptake of cholesterol-containing LDL. LDL-C binds to LDL-R at the plasma membrane then is taken into cells via LDL-R-mediated endocytosis and delivered to sorting endosomes. There a decrease in pH in the endosomal compartment induces the release of LDL-C from LDL-R. Whereas LDL-R is recycled back to the plasma membrane via recycling endosomes, free LDL-C is sent to the lysosomes for degradation and cholesterol release. Finally, LDL particles are degraded and free cholesterol is released into the cytoplasm where cholesterol can enter the membrane of the endoplasmic reticulum and inhibit the synthesis of LDL-R.
Recently, the proprotein convertase subtilisin kexin type 9 (PCSK9) has been identified as a promising drug target since reduced levels of PCSK9 lead to increased LDL-R expression and significant reduction of LDL-C plasma levels and atherosclerotic cardiovascular disease (15, 17, 26, 59). However, the lack of assays to analyze the intracellular membrane trafficking of LDL-R and LDL-C creates significant challenges to the development of therapeutic reagents (31).
Thus, a need exists for a drug screening assay that enables LDL-R and LDL-C trafficking within the cell to be visualized to identify potential cholesterol-lowering therapeutics that regulate plasma cholesterol by mechanisms related to intracellular trafficking of the LDL-R/LDL-C complex and subsequent upregulation of LDL-R expression.